Polypeptide and method of producing IMP using the same

ABSTRACT

The present disclosure relates to a novel polypeptide having an activity of exporting 5′-inosine monophosphate, a microorganism comprising the same, a method for preparing 5′-inosine monophosphate using the same, and a method for increasing export of 5′-inosine monophosphate.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/425,897, filed May 29, 2019, which is a continuationapplication of U.S. application Ser. No. 16/346,041, filed Apr. 29,2019, which is a U.S. national phase application of PCT/KR2018/015937,filed Dec. 14, 2018, which claims priority to KR Application No.10-2017-0173505, filed Dec. 15, 2017. U.S. application Ser. Nos.16/425,897 and 16/346,041 are herein incorporated by reference in theirentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200187_440D1_SEQUENCE LISTING.txt. The text fileis 71 KB, was created on Aug. 29, 2021, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present disclosure relates to a novel protein variant having anactivity of exporting 5′-inosine monophosphate (IMP), a microorganismcomprising the same, and a method for preparing IMP and a method forincreasing export of IMP using the same.

BACKGROUND ART

5′-Inosine monophosphate (hereinafter, IMP), a nucleic acid material, isan intermediate of the nucleic acid metabolism pathway and is used inmany fields such as foods, medicines, various medical applications, etc.In particular, IMP is widely used as an additive for food seasonings orfoods, along with 5′-guanine monophosphate (hereinafter, GMP). AlthoughIMP itself is known to provide a beef taste, it is known to enhance theflavor of monosodium glutamic acid (MSG) and is thus attractingattention as a taste-enhancing nucleic acid-based seasoning.

Examples of methods for producing IMP include a method of enzymaticallydegrading ribonucleic acid extracted from yeast cells (Japanese PatentPublication No. 1614/1957), a method for chemically phosphorylatinginosine produced by fermentation (Agri. Biol. Chem., 36, 1511, etc.), amethod for culturing microorganisms which can directly produce IMP andrecovering IMP in the culture broth, etc. Among these, the method mostfrequently used at present is a method using microorganisms capable ofdirectly producing IMP.

Meanwhile, since enzymes do not always exhibit optimal properties innature with respect to activity, stability, substrate specificity foroptical isomers, etc. required in industrial applications, variousattempts have been made to improve enzymes to suit the intended use bymodification of their amino acid sequences, etc. Among these, althoughrational design and site-directed mutagenesis of enzymes have beenapplied to improve enzyme function, in many cases, these attempts wereshown to be disadvantageous in that information on the structure oftarget enzymes is not sufficient or the structure-function correlationis not clear, thus preventing their effective application. Additionally,a method of improving enzyme activity by attempting the enhancement ofenzymes through directed evolution, which is for screening enzymes ofdesired traits from a library of modified enzymes constructed throughrandom mutagenesis of enzyme genes, was previously reported.

DISCLOSURE Technical Problem

In order to produce IMP in high yield using the method of directlyproducing IMP through microbial fermentation, the IMP should be smoothlyexported. To accomplish such object, the inventors of the presentdisclosure have discovered the protein involved in the activity ofexporting IMP, and also have made many efforts to increase IMPproduction. As a result, they have discovered protein variants havingthe activity of exporting IMP, thereby completing the presentdisclosure.

Technical Solution

An object of the present disclosure is to provide a protein varianthaving the activity of exporting IMP.

Another object of the present disclosure is to provide a polynucleotideencoding the protein variant of the present disclosure.

Still another object of the present disclosure is to provide a vectorincluding the polynucleotide of the present disclosure.

Still another object of the present disclosure is to provide amicroorganism producing IMP, including the protein variant and vector ofthe present disclosure.

Still another object of the present disclosure is to provide a methodfor preparing IMP, including culturing the microorganism of the presentdisclosure in a medium.

Still another object of the present disclosure is to provide a methodfor increasing the export of IMP, including enhancing activity of theprotein variant of the present disclosure, which has the activity ofexporting IMP.

Advantageous Effects of the Invention

IMP can be produced in high yield by culturing a microorganism of thegenus Corynebacterium producing IMP using the protein variant of thepresent disclosure, which is capable of exporting IMP.

BEST MODE FOR CARRYING OUT THE INVENTION

The present disclosure will be described in detail as follows.Meanwhile, each of the explanations and exemplary embodiments disclosedherein can be applied to other respective explanations and exemplaryembodiments. That is, all of the combinations of various factorsdisclosed herein belong to the scope of the present disclosure.Additionally, the scope of the present disclosure should not be limitedby the specific disclosure provided hereinbelow.

To achieve the above objects, an aspect of the present disclosureprovides a protein variant having an activity of exporting IMP.

As used herein, the term “a protein that exports 5′-inosinemonophosphate (IMP)” refers to a protein involved in the extracellularexport of IMP. For the purpose of the present disclosure, the term maybe used interchangeably with a protein having an activity of exportingIMP, an IMP export protein, a protein having an activity of exporting5′-inosine monophosphate, a 5′-inosine monophosphate-exporting protein,etc.; specifically, the protein may be expressed as ImpE, and morespecifically, may be expressed as ImpE1 or ImpE2, but is not limitedthereto. Additionally, the protein may be derived from a microorganismof the genus Corynebacterium, and specifically from Corynebacteriumstationis, but the microorganism is not limited thereto.

The protein, for example, may consist of the amino acid sequencerepresented by SEQ ID NO: 1 or SEQ ID NO: 2, but any sequence having thesame activity as the protein can be included without limitation, and oneof ordinary skill in the art can obtain sequence information fromGenBank of NCBI, a well-known database. Additionally, the protein mayinclude the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or anamino acid sequence having a homology or identity to the sequence of SEQID NO: 1 or SEQ ID NO: 2 of at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%. Additionally, itis obvious that any protein having an amino acid sequence with deletion,modification, substitution, or addition in part of the sequence can alsobe included in the scope of the present disclosure, as long as the aminoacid sequence has a homology or identity described above and has aneffect corresponding to that of the protein.

That is, although described as “a protein having an amino acid sequenceof a particular SEQ ID NO” or “a protein consisting of an amino acidsequence of a particular SEQ ID NO” in the present disclosure, theprotein may have an activity that is identical or corresponding to thatof a protein consisting of an amino acid sequence of the correspondingSEQ ID NO. In such a case, it is obvious that any proteins having anamino acid sequence with deletion, modification, substitution,conservative substitution, or addition in part of the sequence also canbe used in the present disclosure. For example, in the case of havingthe activity that is the same as or corresponding to that of themodified protein, it does not exclude an addition of a sequence upstreamor downstream of the amino acid sequence, which does not alter thefunction of the protein, a mutation that may occur naturally, a silentmutation thereof, or a conservative constitution, and even when thesequence addition or mutation is present, it obviously belongs to thescope of the present disclosure.

As used herein, the term “homology” or “identity” refers to a degree ofmatching with two given amino acid sequences or nucleotide sequences,and may be expressed as a percentage.

The terms “homology” and “identity” may often be used interchangeablywith each other.

The sequence homology or identity of conserved polynucleotide orpolypeptide sequences may be determined by standard alignment algorithmsand can be used with a default gap penalty established by the programbeing used. Substantially homologous or identical sequences aregenerally expected to hybridize under moderate or high stringency, alongthe entire length or at least about 50%, about 60%, about 70%, about80%, or about 90% of the entire length of the sequences. Polynucleotidesthat contain degenerate codons instead of codons in the hybridizingpolypeptides are also considered.

Whether any two polynucleotide or polypeptide sequences have a homology,similarity, or identity may be determined using a known computeralgorithm such as the “FASTA” program (Pearson et al., (1988) [Proc.Natl. Acad. Sci. USA 85]: 2444: using default parameters in 2444).Alternately, it may be determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which isperformed in the Needleman program of the EMBOSS package ((EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277) (version 5.0.0 or versions thereafter) (GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,]Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM JApplied Math 48: 1073). For example, the homology, similarity, oridentity may be determined using BLAST or ClustalW of the NationalCenter for Biotechnology Information (NCBI).

The homology, similarity, or identity of polynucleotide or polypeptidesequences may be determined by comparing sequence information using, forexample, the GAP computer program (e.g., Needleman et al., (1970), J MolBiol. 48: 443) as published (e.g., Smith and Waterman, Adv. Appl. Math(1981) 2:482). In summary, the GAP program defines the homology,similarity, or identity as the value obtained by dividing the number ofsimilarly aligned symbols (i.e., nucleotides or amino acids) into thetotal number of the symbols in the shorter of the two sequences. Defaultparameters for the GAP program may include (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) andthe weighted comparison matrix of Gribskov et al. (1986), Nucl. AcidsRes. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap (or a gap opening penalty of 10and a gap extension penalty of 0.5); and (3) no penalty for end gaps.Accordingly, as used herein, the term “homology” or “identity” refers torelevance between sequences. Specifically, the protein variant of thepresent disclosure having the activity of exporting IMP may be one inwhich at least one amino acid selected from the group consisting of the164^(th) amino acid in the amino acid sequence of SEQ ID NO: 1, the222^(nd) amino acid in the amino acid sequence of SEQ ID NO: 1, the2^(nd) amino acid in the amino acid sequence of SEQ ID NO: 2, and the64^(th) amino acid in the amino acid sequence of SEQ ID NO: 2 issubstituted with another amino acid, but is not limited thereto.

For example, in the protein variant having the activity of exportingIMP, the 164^(th) amino acid in the amino acid sequence of SEQ ID NO: 1is substituted with lysine, arginine, asparagine, glycine, threonine, orproline; the 2^(nd) amino acid in the amino acid sequence of SEQ ID NO:2 is substituted with isoleucine, phenylalanine, methionine, glutamicacid, histidine, or asparagine; or the 64^(th) amino acid in the aminoacid sequence of SEQ ID NO: 2 is substituted with aspartic acid,glutamic acid, asparagine, cysteine, isoleucine, or phenylalanine, butis not limited thereto.

As a specific example, the protein variant having the activity ofexporting IMP may be a protein having the amino acid sequence consistingof SEQ ID NO: 141, 142, 145, 147, 149, or 151, a protein having an aminoacid sequence encoded by the polynucleotide of SEQ ID NO: 153 or 154, ora protein having an amino acid sequence having a homology thereto of atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%. In addition, it is apparent that a proteinhaving a deletion, modification, substitution, or addition of somesequence may be used as the protein of the present disclosure as long asit is a protein having the amino acid sequence with the homology aboveand exhibiting an effect corresponding to that of the protein.

Another aspect of the present disclosure provides a polynucleotideencoding the protein variant, or a vector including the polynucleotide.

As used herein, the term “polynucleotide” refers to a polymer ofnucleotides which is extended in a long chain by covalent bonds and hasa DNA strand or an RNA strand longer than a certain length, and morespecifically, refers to a polynucleotide fragment encoding the proteinvariant.

It is apparent that a polynucleotide, which can be translated by codondegeneracy into a protein consisting of the amino acid sequence of SEQID NO: 141, 142, 145, 147, 149, or 151, a protein consisting of an aminoacid sequence encoded by the polynucleotide of SEQ ID NO: 153 or 154, orinto a protein having a homology thereto, also can be included as thepolynucleotide of the present disclosure. For example, thepolynucleotide of the present disclosure may be a polynucleotide havinga nucleotide sequence of SEQ ID NO: 143, 144, 146, 148, 150, 152, 153,or 154, and more specifically, may be a polynucleotide composed of anucleotide sequence of SEQ ID NO: 143, 144, 146, 148, 150, 152, 153, or154. In addition, a polynucleotide sequence, which encodes a proteinhaving the activity of the protein consisting of an amino acid sequenceof SEQ ID NO: 141, 142, 145, 147, 149, or 151 or an amino acid sequenceencoded by a polynucleotide of SEQ ID NO: 153 or 154 by hybridizationunder stringent conditions with a probe which can be prepared from knowngene sequences, e.g., a complementary sequence to all or part of thenucleotide sequence, may be included without limitation.

The term “stringent conditions” refers to conditions under whichspecific hybridization between polynucleotides is made possible. Suchconditions are specifically described in references (e.g., J. Sambrooket al., supra). For example, the conditions may include performinghybridization between genes having a high homology, a homology of 40% orhigher, specifically 90% or higher, more specifically 95% or higher,even more specifically 97% or higher, and most specifically 99% orhigher, while not performing hybridization between genes having ahomology of lower than the above homologies; or to perform hybridizationonce, specifically two or three times, under conventional washingconditions for southern hybridization of 60° C., 1×SSC, and 0.1% SDS,specifically at a salt concentration and temperature corresponding to60° C., 0.1×SSC, and 0.1% SDS, and more specifically 68° C., 0.1×SSC,and 0.1% SDS.

Hybridization requires that two nucleic acids have a complementarysequence, although mismatches between bases may be possible depending onthe stringency of the hybridization. The term “complementary” is used todescribe the relationship between mutually hybridizable nucleotidebases. For example, with respect to DNA, adenosine is complementary tothymine, and cytosine is complementary to guanine. Accordingly, thepresent disclosure may also include isolated nucleic acid fragmentscomplementary to the entire sequence as well as substantially similarnucleic acid sequences.

Specifically, polynucleotides having a homology can be detected at aT_(m) value of 55° C. using hybridization conditions that include ahybridization step and using the conditions described above.Additionally, the Tm value may be 60° C., 63° C., or 65° C., but is notlimited thereto and may be appropriately adjusted by an ordinary personskilled in the art according to the intended purpose.

The stringency suitable for the hybridization of polynucleotides dependson the length and complementarity of the polynucleotides and the relatedvariables are well known in the art (see Sambrook et al., supra, 9.50 to9.51 and 11.7 to 11.8).

As used herein, the term “vector” refers to a DNA construct includingthe nucleotide sequence of the polynucleotide encoding a target protein,in which the target protein is operably linked to a suitable controlsequence so that the target protein can be expressed in an appropriatehost. The control sequence may include a promoter capable of initiatingtranscription, any operator sequence for controlling the transcription,a sequence encoding an appropriate mRNA ribosome-binding domain, and asequence controlling the termination of transcription and translation.The vector, after being transformed into a suitable host cell, may bereplicated or function irrespective of the host genome, or may beintegrated into the host genome itself.

The vector used in the present disclosure may not be particularlylimited as long as the vector is replicable in the host cell, and it maybe constructed using any vector known in the art. Examples of the vectormay include natural or recombinant plasmids, cosmids, viruses, andbacteriophages. For example, as a phage vector or cosmid vector, pWE15,M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc.may be used; and as a plasmid vector, those based on pBR, pUC,pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used. Specifically, pDZ,pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BACvectors, etc. may be used.

In an embodiment, the polynucleotide encoding the target protein may bereplaced with a modified polynucleotide within the chromosome using avector for the insertion into the chromosome in a cell. The insertion ofthe polynucleotide into the chromosome may be performed using a knownmethod in the art, for example, by homologous recombination, but is notlimited thereto. In particular, a selection marker for confirming theinsertion into the chromosome may be further included. The selectionmarker is used for selection of a transformed cell, i.e., in order toconfirm whether the target nucleic acid has been inserted, and markerscapable of providing selectable phenotypes such as drug resistance,nutrient requirement, resistance to cytotoxic agents, and expression ofsurface proteins may be used. Under the circumstances where selectiveagents are treated, only the cells capable of expressing the selectionmarkers can survive or express other phenotypic traits, and thus thetransformed cells can be easily selected.

Still another aspect of the present disclosure provides a microorganismproducing IMP, including the protein variant of the present disclosure,the polynucleotide of the present disclosure encoding the proteinvariant, or the vector of the present disclosure. Specifically, themicroorganism including the protein variant and/or a polynucleotideencoding the protein variant may be a microorganism prepared bytransformation using a vector containing the polynucleotide encoding theprotein variant, but the microorganism is not limited thereto.

As used herein, the term “transformation” refers to a process ofintroducing a vector including a polynucleotide encoding a targetprotein into a host cell, thereby enabling the expression of the proteinencoded by the polynucleotide in the host cell. For the transformedpolynucleotide, it does not matter whether it is inserted into thechromosome of the host cell and located therein or located outside thechromosome, as long as the transformed polynucleotide can be expressedin the host cell. Additionally, the polynucleotide includes DNA and RNAwhich encode the target protein. The polynucleotide may be inserted inany form as long as it can be introduced into a host cell and expressedtherein. For example, the polynucleotide may be introduced into a hostcell in the form of an expression cassette, which is a gene constructincluding all of the essential elements required for self-expression.The expression cassette may conventionally include a promoter operablylinked to the polynucleotide, a transcription termination signal, aribosome-binding domain, and a translation termination signal. Theexpression cassette may be in the form of a self-replicable expressionvector. Additionally, the polynucleotide may be introduced into a hostcell as is and operably linked to a sequence essential for itsexpression in the host cell, but is not limited thereto.

Additionally, as used herein, the term “operably linked” refers to afunctional linkage between a promoter sequence, which initiates andmediates the transcription of the polynucleotide encoding the targetprotein, i.e., a conjugate of the present disclosure, and the above genesequence.

As used herein, the term “IMP-producing microorganism” refers to amicroorganism which is naturally capable of producing IMP; or amicroorganism introduced an ability to produce or export IMP to whoseparent strain is not naturally capable of producing and/or exporting IMPwhich is In the present disclosure, the microorganism producing IMP canbe used interchangeably with a microorganism having an activity ofexporting IMP.

The IMP-producing microorganism is a cell or microorganism whichincludes a protein variant having an activity of exporting IMP or apolynucleotide encoding the protein variant, or which is transformedwith a vector containing the polynucleotide encoding the proteinvariant, and is thereby capable of expressing the protein variant. Forthe purposes of the present disclosure, the host cell of theIMP-producing microorganism or microorganism may be any microorganismincluding the protein variant thus capable of producing IMP. Forexample, the microorganism may be a microorganism of the genusEscherichia, a microorganism of the genus Serratia, a microorganism ofthe genus Erwinia, a microorganism of the genus Enterobacteria, amicroorganism of the genus Salmonella, a microorganism of the genusStreptomyces, a microorganism of the genus Pseudomonas, a microorganismof the genus Brevibacterium, a microorganism of the genusCorynebacterium, etc., and specifically, a microorganism of the genusCorynebacterium.

As used herein, the term “IMP-producing microorganism of the genusCorynebacterium” refers to a microorganism of the genus Corynebacteriumwhich is naturally capable of producing IMP or capable of producing IMPby modification. Specifically, as used herein, the microorganism of thegenus Corynebacterium capable of producing IMP refers to a native strainof the microorganism of the genus Corynebacterium capable of producingIMP; or a microorganism of the genus Corynebacterium with enhancedabilities to produce IMP prepared by inserting a gene associated withIMP production or by enhancing or attenuating the endogenous geneassociated with IMP production. More specifically, in the presentdisclosure, the microorganism of the genus Corynebacterium capable ofproducing IMP refers to a microorganism of the genus Corynebacteriumwhich has improved abilities to produce IMP by including a proteinvariant having an activity of exporting IMP or a polynucleotide encodingthe protein variant, or by being transformed with a vector containingthe polynucleotide encoding the protein variant. The “microorganism ofthe genus Corynebacterium with enhanced abilities to produce IMP” refersto a microorganism of the genus Corynebacterium with improved abilitiesto produce IMP compared to that of its parent strain beforetransformation or that of an unmodified microorganism of the genusCorynebacterium. The “unmodified microorganism of the genusCorynebacterium” refers to a native type of the microorganism of thegenus Corynebacterium, a microorganism of the genus Corynebacteriumwhich does not contain a protein variant capable of exporting IMP, or amicroorganism of the genus Corynebacterium which is not transformed witha vector containing a polynucleotide encoding the protein variantcapable of exporting IMP.

In an embodiment of the present disclosure, the microorganism of thepresent disclosure may be a microorganism of the genus Corynebacterium,in which the activity of adenylosuccinate synthetase and/or IMPdehydrogenase is further attenuated.

In the present disclosure, “a microorganism of the genusCorynebacterium” specifically refers to Corynebacterium glutamicum,Corynebacterium ammoniagenes, Brevibacterium lactofermentum,Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacteriumefficiens, Corynebacterium stationis, etc., but the microorganism is notnecessarily limited thereto.

Still another aspect of the present disclosure provides a method forpreparing IMP, including culturing the microorganism of the genusCorynebacterium in a medium.

Specifically, the method of the present disclosure may additionallyinclude a step of recovering IMP from the microorganism or medium.

In the above method, the cultivation of the microorganism may beperformed in a batch process, continuous process, fed-batch process,etc. known in the art, but the cultivation process is not particularlylimited thereto. In particular, with respect to the cultivationconditions, the pH of the culture may be adjusted to a suitable pH(e.g., pH 5 to 9, specifically pH 6 to 8, and most specifically with anappropriate basic compound (e.g., sodium hydroxide, potassium hydroxide,or ammonia) or acidic compound (e.g., phosphoric acid or sulfuric acid),and the aerobic condition of the culture may be maintained byintroducing oxygen or an oxygen-containing gas mixture to the culture.The cultivation temperature may generally be in the range of 20° C. to45° C., and specifically 25° C. to 40° C. for about 10 to 160 hours, butthe cultivation conditions are not limited thereto. The IMP produced bythe above cultivation may be secreted into the culture or may beretained in the cells.

Additionally, examples of the carbon sources to be used in the culturemedium may include sugars and carbohydrates (e.g., glucose, sucrose,lactose, fructose, maltose, molasses, starch, and cellulose); oils andfats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil);fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid);alcohols (e.g., glycerol and ethanol); and organic acids (e.g., aceticacid), but are not limited thereto. These carbon sources may be usedalone or in combination, but are not limited thereto. Examples of thenitrogen sources to be used in the culture medium may includenitrogen-containing organic compounds (e.g., peptone, yeast extract,meat gravy, malt extract, corn steep liquor, soybean flour, and urea) orinorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammoniumphosphate, ammonium carbonate, and ammonium nitrate), etc. Thesenitrogen sources may be used alone or in combination, but are notlimited thereto. Examples of the phosphorus sources to be used in theculture medium may include potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, corresponding sodium-containing salts, etc., but arenot limited thereto. Additionally, metal salts (e.g., magnesium sulfateor iron sulfate), amino acids, vitamins, etc., which are essentialgrowth-promoting materials, may be contained in the medium.

In the present disclosure, the method for recovering the IMP produced inthe step of cultivation may be performed by collecting the IMP from theculture broth using an appropriate method known in the art. For example,methods such as centrifugation, filtration, anion exchangechromatography, crystallization, HPLC, etc. may be used, and the desiredIMP can be recovered from a culture or cultured microorganism using anappropriate method known in the art.

Further, the recovery may include a purification process and may beperformed using an appropriate method known in the art. Thus, the IMP tobe recovered may be in a purified form or a microorganism fermentationbroth containing IMP.

Still another aspect of the present disclosure provides a compositionfor producing IMP, including the protein variant of the presentdisclosure, which has the activity of exporting IMP, or a polynucleotideencoding the same.

The composition of the present disclosure may further include, withoutlimitation, a constitution capable of operating the polynucleotide. Inthe composition of the present disclosure, the polynucleotide may be ina form included within a vector to express an operably linked gene inthe introduced host cell.

Additionally, the composition may further include any suitableexcipients conventionally used in the composition for producing IMP.Such excipients may be, for example, preservatives, humectants,suspending agents, buffers, stabilizing agents, or isotonic agents, butare not limited thereto.

Still another aspect of the present disclosure provides use of theprotein of the present disclosure for increasing the production of IMPin the microorganism of the genus Corynebacterium.

Still another aspect of the present disclosure provides a method forincreasing the export of IMP, including enhancing the activity of theprotein variant, which has the activity of exporting IMP, in themicroorganism of the genus Corynebacterium.

The terms “protein having the activity of exporting IMP”, “enhancement”,and “microorganism of the genus Corynebacterium” are as described above.

Still another aspect of the present disclosure provides use of theprotein of the present disclosure for increasing the export of IMP inthe microorganism of the genus Corynebacterium.

DETAILED DESCRIPTION OF THE EMBODIMENT

Hereinafter, the present disclosure will be described in detail throughexemplary embodiments. However, it should be obvious to one of ordinaryskill in the art that these exemplary embodiments are provided for thepurpose of illustration only and are not intended to limit the scope ofthe present disclosure.

Example 1: Discovery of IMP Export Proteins

A genomic DNA library of Corynebacterium stationis ATCC6872 was preparedfor the identification of membrane proteins of Corynebacterium involvedin the export of IMP. Then, since the wild-type strain ofCorynebacterium cannot produce IMP, or even if it does produce IMP, itproduces only a small amount thereof, a strain called CJI0323, which iscapable of producing IMP, derived from the ATCC6872 strain was preparedfor the identification of the ability to produce IMP. The CJI0323 strainprepared was subjected to screening of membrane proteins involved in IMPexport using the genomic DNA library of the ATCC6872 strain. Thespecific details of the experiment are as follows.

Example 1-1: Selection of IMP-Producing Strain, CJI0323

The ATCC6872 cells were suspended in a phosphate buffer (pH 7.0) orcitrate buffer (pH 5.5) at a concentration of 10⁷ cells/mL to 10⁸cells/mL to prepare an ATCC6872-derived IMP-producing strain, and thecells were subjected to UV treatment to induce mutation. The resultingcells were washed twice with a 0.85% saline solution, and then dilutedand plated on a medium, which was prepared by adding aresistance-providing material at an appropriate concentration to aminimal medium containing 1.7% agar, and colonies were obtainedthereafter. Each colony was cultured in a nutrient medium and culturedin a seed medium for 24 hours. After culturing the colonies for 3 to 4days in a fermentation medium, the colony with the highest abilities toproduce IMP accumulated in the culture medium was selected. In thecourse of preparing a strain capable of producing IMP at highconcentration, in order to provide adenine auxotrophy, guanine leakage,lysozyme susceptibility, 3,4-dihydroproline resistance, streptomycinresistance, azetidine carboxylic acid resistance, thiaprolineresistance, azaserine resistance, sulfaguanidine resistance, norvalineresistance, and trimethoprim resistance, the procedures above wereperformed sequentially for each material. As a result, CJI0323, whichshowed resistance to the above materials and excellent abilities toproduce IMP, was finally selected. The degree of resistance betweenATCC6872 and CJI0323 was compared and the results are shown in Table 1below.

TABLE 1 Characteristics ATCC6872 CJI0323 Adenine auxotrophyNon-auxotrophy Auxotrophy Guanine leakage Non-auxotrophy Leakyauxotrophy Lysozyme susceptibility 80 μg/mL 8 μg/mL 3,4-Dihydroprolineresistance 1000 μg/mL 3500 μg/mL Streptomycin resistance 500 μg/mL 2000μg/mL Azetidine carboxylic acid 5 mg/mL 30 mg/mL resistance Thiaprolineresistance 10 μg/mL 100 μg/mL Azaserine resistance 25 μg/mL 100 μg/mLSulfaguanidine resistance 50 μg/mL 200 μg/mL Norvaline resistance 0.2mg/mL 2 mg/mL Trimethoprim resistance 20 μg/mL 100 μg/mL

-   -   Minimal medium: 2% glucose, 0.3% sodium sulfate, 0.1% KH₂SO₄,        0.3% K₂HPO₄, 0.3% magnesium sulfate, calcium chloride (10 mg/L),        iron sulfate (10 mg/L), zinc sulfate (1 mg/L), manganese        chloride (3.6 mg/L), L-cysteine (20 mg/L), calcium pantothenate        (10 mg/L), thiamine hydrochloride (5 mg/L), biotin (30 μg/L),        adenine (20 mg/L), guanine (20 mg/L), pH 7.3    -   Nutrient medium: 1% peptone, 1% meat juice, 0.25% sodium        chloride, 1% yeast extract, 2% agar, pH 7.2    -   Seed medium: 1% glucose, 1% peptone, 1% meat juice, 1% yeast        extract, 0.25% sodium chloride, adenine (100 mg/L), guanine (100        mg/L), pH 7.5    -   Fermentation medium: 0.1% sodium glutamate, 1% ammonium        chloride, 1.2% magnesium sulfate, 0.01% calcium chloride, iron        sulfate (20 mg/L), manganese sulfate (20 mg/L), zinc sulfate (20        mg/L), copper sulfate (5 mg/L), L-cysteine (23 mg/L), alanine        (24 mg/L), nicotinic acid (8 mg/L), biotin (45 μg/L), thiamine        hydrochloride (5 mg/L), adenine (30 mg/L), 1.9% phosphoric acid        (85%), 2.55% glucose, 1.45% fructose

Example 1-2: Experiments on Fermentation Titer of CJI0323

The seed medium (2 mL) was dispensed into test tubes (diameter: 18 mm),which were then autoclaved and each inoculated with ATCC6872 andCJI0323. Thereafter, the resultants were shake-cultured at 30° C. for 24hours and then used as a seed culture solution. The fermentation medium(29 mL) was dispensed into Erlenmeyer flasks (250 mL) for shaking,autoclaved at 121° C. for 15 minutes, and the seed culture solution (2mL) was inoculated thereto and cultured for 3 days. The cultureconditions were set to 170 rpm, 30° C., and a pH of 7.5.

Upon completion of the culture, the amount of IMP produced was measuredby HPLC (SHIMAZDU LC20A) and the results of the culture are shown inTable 2 below.

TABLE 2 Strain IMP (g/L) ATCC6872 0 CJI0323 9.52

The CJI0323 strain was named as Corynebacterium stationis CN01-0323. Thestrain was deposited under the Budapest Treaty to the Korean CultureCenter of Microorganisms (KCCM) on Nov. 7, 2017. In addition, the strainwas designated as Accession No. KCCM12151P.

Example 1-3: Discovery of Exporting Proteins

Screening conditions showing growth inhibition of the CJI0323 strainwere established by additionally adding IMP to the minimal mediumcontaining 1.7% agar. The plasmids of the genomic library of theATCC6872 strain were transformed into the CJI0323 strain byelectroporation (van der Rest et al. 1999), and those colonies in whichthe growth inhibition was released under the medium conditionssupplemented with an excess amount of IMP were selected. Plasmids wereobtained from the selected colonies and analyzed by a sequencingtechnique. As a result, one kind of membrane protein involved in therelease of the growth inhibition was identified under the conditionwhere an excess amount of IMP was added.

The one kind of membrane protein from Corynebacterium was identifiedbased on the amino acid sequence of SEQ ID NO: 2 and the nucleotidesequence of SEQ ID NO: 4 (NCBI GenBank: NZ_CP014279, WP_066795121, MFStransporter). The membrane protein is known as the MFS transporter, butits specific function has not been confirmed, and further, its functionregarding the IMP export is still unknown. In the present disclosure,the membrane protein was named ImpE2(WT).

Example 2: Identification of ImpE1 and ImpE2 Example 2-1: Confirmationof impE1 and impE2

In order to examine the functions of the membrane protein, ImpE2, thegene structure of SEQ ID NO: 4 was confirmed in the NCBI (NCBI GenBank:NZ_CP014279, WP_066795121, MFS transporter). As a result, it wasconfirmed that the 7 bp starting portion of the ORF of SEQ ID NO: 4(impE2) overlaps in 7 bp with a different gene (NCBI GenBank:NZ_CP014279, WP_066795119, transcriptional regulator), which is locatedupstream of impE2. Since the functions of the gene located upstream ofimpE2 and the protein encoded by the gene have not been confirmed, inthe present disclosure, the protein was named ImpE1(WT) (the amino acidsequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 3).

Example 2-2: Preparation of impE1- or impE2-Deficient Vector

In order to confirm whether the deletion of ImpE1 or ImpE2, which areinvolved in releasing the growth inhibition caused by IMP as identifiedin Examples 1 and 2-1, in an IMP-producing strain can reduce itsIMP-exporting ability, attempts were made to prepare vectors deficientin each of the genes.

The gene fragments for preparing the vectors were obtained by PCR usingthe genomic DNA of the ATCC6872 strain as a template.

Specifically, the PCR for impE1 was performed using primers of SEQ IDNOS: 5 and 6 and primers of SEQ ID NOS: 7 and 8; and the PCR for impE2was performed using the primers of SEQ ID NOS: 9 and 10 and primers ofSEQ ID NOS: 11 and 12 (Table 3).

TABLE 3 SEQ ID NO Primer Sequence (5′ to 3′) 5 impE1 kop-1GCTCTAGACGAGAAAGCTAAAGCCGGTGA 6 impE1 kop-2GTTTTTAGCTACCATTGTTACACCCCGTG CAAGTTT 7 impE1 kop-3GCACGGGGTGTAACAATGGTAGCTAAAAA CTCCACC 8 impE1 kop-4GCTCTAGAAATAGTTGGGGAAGTCCACTC 9 impE2 kop-1GCTCTAGACTTGGATGACCTGGTGGAAAA 10 impE2 kop-2CTTGGAGAAAATTTCCTACCATTCCAGTC CTTTCGT 11 impE2 kop-3GGACTGGAATGGTAGGAAATTTTCTCCAA GGGAAAT 12 impE2 kop-4GGACTAGTGGATTGTGTTGACGCACGATG 13 impE1E2kop-2CTTGGAGAAAATTTCTGTTACACCCCGTG CAAGTTT 14 impE1E2kop-3GCACGGGGTGTAACAGAAATTTTCTCCAA GGGAAAT

In particular, the primers used were prepared based on information on agene of Corynebacterium stationis (ATCC687 2) (NCBI Genbank:NZ_CP014279) registered in NIH GenBank and the nucleotide sequencesadjacent thereto.

PCR was performed by initial denaturation at 94° C. for 5 minutes; 25cycles consisting of denaturation at 94° C. for 30 seconds, annealing at52° C. for 30 minutes, and polymerization at 72° C. for 1 minute; andfinal polymerization at 72° C. for 5 minutes.

Overlapping PCR was performed using two fragments of the impE1 gene,which were amplified using the primers of SEQ ID NOS: 5 and 6 and theprimers of SEQ ID NOS: 7 and 8, as templates, and as a result, apolynucleotide template (1.8 kbp) was obtained. The obtained genefragment was cloned into a linearized pDZ vector (Korean Patent No.10-0924065 and International Patent Publication No. 2008-033001), whichwas digested with the restriction enzyme (XbaI), and ligated using T4ligase, and thereby the pDZ-ΔimpE1 vector was prepared. Additionally,overlapping polymerase chain reaction was performed using a fragment ofthe impE2 gene, amplified using the primers of SEQ ID NOS: 9 and 10, andtwo fragments of the impE2 gene, amplified using the primers of SEQ IDNOS: 11 and 12, as templates, and as a result, a polynucleotide template(1.7 kbp) was obtained. The obtained gene fragment was digested withrestriction enzymes, XbaI and SpeI. The gene fragment was cloned usingT4 ligase into a linearized pDZ vector, which had already been digestedwith the restriction enzyme (XbaI), and thereby the pDZ-ΔimpE2 vectorwas prepared.

Example 2-3: Preparation of impE1- and impE2-Integration-DeficientVectors

Since the impE1 and impE2 genes, which encode proteins involved inreleasing the growth inhibition caused by IMP, are overlapped, there isa need to regulate both genes simultaneously. Therefore, attempts weremade to prepare a vector in which both impE1 and impE2 are deficient.

For the PCR of impE1 and impE2 genes, primers of SEQ ID NOS: 5 and 13and primers of SEQ ID NOS: 14 and 12 were used. The primers used wereprepared based on information on a gene of Corynebacterium stationis(ATCC6872) (NCBI Genbank: NZ_CP014279) registered in NIH GenBank and thenucleotide sequences adjacent thereto. Overlapping PCR was performedusing a fragment of the impE1 gene, amplified using the primers of SEQID NOS: 5 and 13, and two fragments of the impE2 gene, amplified usingthe primers of SEQ ID NOS: 14 and 12, as templates, and as a result, apolynucleotide template (2.0 kbp) was obtained. The obtained genefragments were digested with XbaI and SpeI, respectively. The genefragments were cloned using T4 ligase into a linearized pDZ vector,which had already been digested with the restriction enzyme (XbaI), andthereby the pDZ-ΔimpE1E2 vector was prepared.

Example 2-4: Preparation of impE1- and impE2-Deficient Strains

The two kinds of plasmids prepared in Example 2-2 and one kind ofplasmid prepared in Example 2-3 were each transformed into the CJI0323strain by electroporation (using the transformation method disclosed inAppl. Microbiol. Biotechnol. (1999) 52: 541 to 545). The strains inwhich the vector was inserted into the chromosome by recombination ofthe homologous sequences were selected on a medium containing kanamycin(25 mg/L). The selected primary strains were subjected to a secondcross-over. The genetic deficiency in the finally transformed strainswas confirmed by performing PCR using the primer pairs of SEQ ID NOS: 5and 8, SEQ ID NOS: 9 and 12, and SEQ ID NOS: 5 and 12.

The selected strains were named CJI0323_ΔimpE1, CJI0323_ΔimpE2, andCJI0323_ΔimpE1E2. Additionally, the abilities to produce IMP of thesestrains was evaluated.

The seed medium (2 mL) was dispensed into test tubes (diameter: 18 mm),which were then autoclaved, each inoculated with CJI0323,CJI0323_ΔimpE1, CJI0323_ΔimpE2, and CJI0323_ΔimpE1E2, shake-cultured at30° C. for 24 hours, and used as seed culture solutions. Thefermentation medium (29 mL) was dispensed into Erlenmeyer flasks (250mL) for shaking and autoclaved at 121° C. for 15 minutes. Then, the seedculture solution (2 mL) was inoculated thereto and the resultant wascultured for 3 days. The culture conditions were set to 170 rpm, 30° C.,and a pH of 7.5.

Upon completion of the culture, the amount of IMP produced was measuredby HPLC, and the results of the culture are shown in Table 4 below.

TABLE 4 Strain IMP (g/L) CJI0323 9.52 CJI0323_ΔimpE1 1.92 CJI0323_ΔimpE21.88 CJI0323_ΔimpE1E2 1.80

The IMP amount accumulated in each strain was compared with that of theparent strain, Corynebacterium stationis CJI0323. As a result, it wasfound that, as shown in Table 4 above, the IMP concentrations of thestrains CJI0323_ΔimpE1, CJI0323_ΔimpE2, and CJI0323_ΔimpE1E2 werereduced by about 8 g/L under the same conditions compared to the parentstrain, confirming that ImpE1 and ImpE2 are proteins involved in the IMPexport.

Example 3: Confirmation of Nucleotide Sequences of impE1 and impE2 ofIMP-Producing Strain, CJI0323

In the case of the CJI0323 strain producing IMP at high concentration inExample 1, it is possible that the strain has an improved IMP-exportingability so as to produce IMP at high concentration. Accordingly, anattempt was made to confirm the presence of any mutation in impE1 andimpE2 of the CJI0323 strain.

The chromosomal DNA of the CJI0323 strain was amplified by polymerasechain reaction (hereinafter, “PCR”). Specifically, first, PCR wasperformed by repeating 28 cycles consisting of denaturation at 94° C.for 1 minute, annealing at 58° C. for 30 seconds, and polymerization at72° C. for 2 minutes using the chromosomal DNA of the CJI0323 strain asa template along with the primers of SEQ ID NOS: 15 and 16 (Table 5),and thereby a fragment of about 2.8 kbp was amplified.

TABLE 5 SEQ ID NO Primer Sequence (5′ to 3′) 15 impE1E2 seqFGAACGGAGTCATCTCCTTTGC 16 impE1E2 seqR CCAAACGCTCTGCAAGAAACTG

Upon analysis of the nucleotide sequence using the same primers, it wasconfirmed that the 490^(th) nucleotide of the impE1 gene (i.e., g) wassubstituted with ‘a’, compared to the nucleotide sequence of thewild-type strain, ATCC6872. This substitution indicates that there was amodification in which the 164^(th) amino acid of the ImpE1 protein(i.e., glutamic acid) was substituted with lysine.

Additionally, it was confirmed that the 4^(th) nucleotide of the impE2gene (i.e., g) was substituted with ‘a’ (this means that the 666^(th)nucleotide of the impE1 gene (i.e., g) was substituted with ‘a’) and the191^(st) nucleotide of the impE1 gene (i.e., g) was substituted with‘a’. These substitutions indicate that there were modifications in whichthe 2^(nd) amino acid of the ImpE2 protein (i.e., valine), whichcorresponds to the 222^(nd) amino acid of the ImpE1 protein, wassubstituted with isoleucine; and the 64^(th) amino acid of the ImpE2protein (i.e., glycine) was substituted with glutamic acid.

The impE1 nucleotide of the CJI0323 strain was named impE1_CJI0323 (SEQID NO: 143) and the protein thereof was named ImpE1_CJI0323 (SEQ ID NO:141), whereas the impE2 nucleotide of the CJI0323 strain was namedimpE2_CJI0323 (SEQ ID NO: 144) and the protein thereof was namedImpE2_CJI0323 (SEQ ID NO: 142).

Example 4: Recovery of Modifications in impE1 and impE2 Example 4-1:Preparation of Vectors for Recovering Modifications in impE1 or impE2

In Example 3, the presence of any modification in impE1 and impE2 of theIMP-producing strain CJI0323 was examined. As a result, it was confirmedthat impE1 had one modification and impE2 had two modifications. Sincethe CJI0323 strain produces IMP at a high concentration, it is highlylikely that the modification is one that can improve the ability toexport IMP. Accordingly, after recovering the mutated impE1 and impE2 tothe native wild-type ImpE without modification, the following experimentwas performed to confirm whether each modification actually imparted theIMP-exporting ability.

To prepare a recovery vector, PCR was performed using Corynebacteriumstationis ATCC6872 as a template.

The impE1impE2 gene fragment amplified using the primers of SEQ ID NOS:17 and 18 was treated with a restriction enzyme, XbaI, and cloned intothe XbaI restriction site on the pDZ vector, and thereby thepDZ-impE1E2(WT) was prepared.

Example 4-2: Preparation of Vectors with Single Modification in impE1 orimpE2

A vector with a single E164K modification in the ImpE1 gene was preparedusing the native wild-type strain, Corynebacterium stationis ATCC6872,as a template along with the primers of SEQ ID NOS: 19 and 20 andprimers of SEQ ID NOS: 21 and 22. Overlapping PCR was performed using anE164K-1 gene fragment amplified using the primers of SEQ ID NOS: 19 and20 and two E164K-2 gene fragments amplified using the primers of SEQ IDNOS: 21 and 22, and thereby a template with a 1.8 kbp polynucleotide wasobtained. The obtained gene fragments were digested with XbaI and clonedinto a linearized pDZ vector, which had already been digested with XbaI,using T4 ligase, and thereby the pDZ-impE1(E164K) vector was prepared.

A vector with a single V2I modification in the ImpE2 gene was preparedusing the ATCC6872 strain as a template along with the primers of SEQ IDNOS: 19 and 23 and primers of SEQ ID NOS: 24 and 22. Overlapping PCR wasperformed using a V2I-1 gene fragment amplified using the primers of SEQID NOS: 19 and 23 and two V2I-2 gene fragments amplified using theprimers of SEQ ID NOS: 24 and 22, and thereby a template with a 1.8 kbppolynucleotide was obtained. The obtained gene fragments were digestedwith XbaI and cloned into a linearized pDZ vector, which had alreadybeen digested with XbaI, using T4 ligase, and thereby the pDZ-impE2(V2I)vector was prepared.

A vector with a single G64E modification in the ImpE2 gene was preparedusing the ATCC6872 strain as a template along with the primers of SEQ IDNOS: 19 and 25 and primers of SEQ ID NOS: 26 and 22. Overlapping PCR wasperformed using a G64E-1 gene fragment amplified using the primers ofSEQ ID NOS: 19 and 25 and two G64E-2 gene fragments amplified using theprimers of SEQ ID NOS: 26 and 22, and thereby a template with a 1.8 kbppolynucleotide was obtained. The obtained gene fragments were digestedwith XbaI and cloned into a linearized pDZ vector, which had alreadybeen digested with XbaI, using T4 ligase, and thereby thepDZ-impE2(G64E) vector was prepared.

TABLE 6 SEQ ID NO Primer Sequence (5′ to 3′) 17 impE1E2 WT FGCTCTAGAGAACGGAGTCATCTCCTTTGC 18 impE1E2 WT RGCTCTAGACCAAACGCTCTGCAAGAAACTG 19 impE1 164K-1GCTCTAGACTTGGATGACCTGGTGGAAAA 20 impE1 164K-2CTGGGGCGCGTTGTTTTTCAGGATGCTCCC GAAGACG 21 impE1 164K-3AACAACGCGCCCCAGAATTGG 22 impE1 164K-4 GCTCTAGAAATAGTTGGGGAAGTCCACTC 23impE2 V2I-2 TGGAGTTTTTAGCTATCATTCCAGTCCTT TCGTGTAA 24 impE2 V2I-3TAGCTAAAAACTCCACCCCAA 25 impE2 G64E-2 CCGAAAATCATCTGCTCCAAAGAGCTCATCAGCATGG 26 impE2 G64E-3 GCAGATGATTTTCGGTTCCGC

Example 4-3: Recovery of impE1, impE2 Modifications and Preparation ofStrains with Single Modification

The plasmid prepared in Example 4-1 was transformed into the CJI0323strain by electroporation (using the transformation method disclosed inAppl. Microbiol. Biotechnol. (1999) 52: 541 to 545). The strains inwhich the vector was inserted into the chromosome by recombination ofthe homologous sequences were selected on a medium containing kanamycin(25 mg/L). The selected primary strains were subjected to a secondcross-over. The recovery of the modification in the finally transformedstrains was confirmed by performing PCR using the primer pair of SEQ IDNOS: 15 and 16, followed by nucleotide sequencing analysis. The preparedstrain was named CJI0323_impE1E2(W7).

The three kinds of plasmids prepared in Example 4-2 were eachtransformed into the CJI0323_impE1E2(W7) strain by electroporation(using the transformation method disclosed in Appl. Microbiol.Biotechnol. (1999) 52: 541 to 545). The strains in which the vector wasinserted into the chromosome by recombination of the homologoussequences were selected on a medium containing kanamycin (25 mg/L). Theselected primary strains were subjected to a second cross-over. Theintroduction of the modification in the finally transformed strains wasconfirmed by performing PCR using the primer pair of SEQ ID NOS: 15 and16, followed by nucleotide sequencing analysis. The selected strainswere named CJI0323_impE1(E164K), CJI0323_impE2(V2I), andCJI0323_impE2(G64E).

The Corynebacterium stationis CJI0323_impE1(E164K), Corynebacteriumstationis CJI0323_impE2(V2I), and Corynebacterium stationisCJI0323_impE2(G64E) strains were deposited under the Budapest Treaty tothe Korean Culture Center of Microorganisms (KCCM) on Nov. 2, 2018. Inaddition, the strains were designated with Accession Nos. KCCM12359P,KCCM12360P, and KCCM12361P, respectively.

Example 4-4: Preparation of impE1- and impE2-Integration-ModifiedStrains

The pDZ-impE2(V2I) and pDZ-impE2(G64E) plasmids prepared in Example 4-2were transformed into the CJI0323_impE1(E164K) strain by electroporation(using the transformation method disclosed in Appl. Microbiol.Biotechnol.(1999) 52: 541 to 545). The strains in which the vectors wereinserted into the chromosome by recombination of the homologoussequences were selected on a medium containing kanamycin (25 mg/L). Theselected primary strains were subjected to a second cross-over. Theintroduction of the modification in the finally transformed strains wasconfirmed by performing PCR using the primer pair of SEQ ID NOS: 15 and16, followed by nucleotide sequencing analysis. The prepared strainswere named CJI0323_impE1(E164K)_impE2(V2I) and CJI0323_impE1(164K)impE2(G64E).

The pDZ-impE2(G64E) plasmid was transformed into the CJI0323_impE2(V2I)strain by electroporation (using the transformation method disclosed inAppl. Microbiol. Biotechnol. (1999) 52: 541 to 545). The strains inwhich the vector was inserted into the chromosome by recombination ofthe homologous sequences were selected on a medium containing kanamycin(25 mg/L). The selected primary strains were subjected to a secondcross-over. The introduction of the modification in the finallytransformed strains was confirmed by performing PCR using the primerpair of SEQ ID NOS: 15 and 16, followed by nucleotide sequencinganalysis. The selected strain was named CJI0323_impE2(V2I)(G64E).

Example 4-5: Evaluation of Strains with impE1, impE2 Modifications

The seed medium (2 mL) was dispensed into test tubes (diameter: 18 mm),which were then autoclaved, each inoculated with CJ10323_impE1E2(W1),CJ10323_impE1(E164K), CJ10323_impE2(V2I), CJ10323_impE2(G64E),CJ10323_impE1(E164K)_impE2(V2I), CJ10323_impE1(E164K)_impE2(G64E), andCJ10323_impE2(V2I)(G64E), shake-cultured at 30° C. for 24 hours, andused as seed culture solutions. The fermentation medium (29 mL) wasdispensed into Erlenmeyer flasks (250 mL) for shaking and autoclaved at121° C. for 15 minutes. Then, the seed culture solutions (2 mL) wereinoculated thereto and the resultants were cultured for 3 days. Theculture conditions were set to 170 rpm, 30° C., and a pH of 7.5.

Upon completion of the culture, the amount of IMP produced was measuredby HPLC, and the results of the culture are shown in Table 7 below.

TABLE 7 Strain IMP (g/L) CJI0323 9.52 CJI0323_impE1E2(WT) 2.32CJI0323_impE1(E164K) 2.57 CJI0323_impE2(V2I) 3.11 CJI0323_impE2(G64E)3.27 CJI0323_impE1(E164K)_impE2(V2I) 4.24CJI0323_impE1(E164K)_impE2(G64E) 6.27 CJI0323_impE2(V2I)(G64E) 7.35

As shown above, it was confirmed that with respect to each modificationposition, one kind of modification, the integration of two kinds ofmodifications, and the integration of three kinds of modifications wereall involved in the IMP export. Accordingly, in a microorganism of thegenus Corynebacterium which does not produce IMP or produces only asmall amount thereof, the increase in the amount of IMP production dueto modifications of the ImpE protein of the present disclosure can beinterpreted to be very meaningful.

Example 5: Substitution of Amino Acids in impE1, impE2 Modificationswith Another Amino Acids Example 5-1: Preparation of vectors forsubstitutional insertion of amino acids in impE1, impE2 modifications

To confirm the positional importance of the representative three kindsof modifications (i.e., impE1(E164K), impE2(V2I), and impE2(G64E)) withenhanced abilities to produce IMP as identified in the results above, avector for introducing modifications (e.g., a modification ofsubstituting the 164^(th) amino acid in the amino acid sequence ofimpE1, the 2^(nd) amino acid in the amino acid sequence of impE2, andthe 64^(th) amino acid in the amino acid sequence of impE2 with ananother amino acid) was prepared.

Firstly, the procedure of preparing the vector for the introduction ofthe ImpE1(E164K) modification is as follows.

Based on the reported polynucleotide sequences, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template along with primer pairs between theprimer of SEQ ID NO: 27 and each of SEQ ID NOS: 28 to 45. PCR wasperformed by initial denaturation at 94° C. for 5 minutes; 20 cyclesconsisting of denaturation at 94° C. for 30 seconds, annealing at 55° C.for 30 seconds, and polymerization at 72° C. for 1 minute; and finalpolymerization at 72° C. for 5 minutes. As a result, 18 kinds of 0.7 kbppolynucleotides were obtained. Then, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template along with primer pairs between theprimer of SEQ ID NO: 46 and each of SEQ ID NOS: 47 to 64. PCR wasperformed by initial denaturation at 94° C. for 5 minutes; 20 cyclesconsisting of denaturation at 94° C. for 30 seconds, annealing at 55° C.for 30 seconds, and polymerization at 72° C. for 1 minute; and finalpolymerization at 72° C. for 5 minutes. As a result, 18 kinds of 0.7 kbppolynucleotides were obtained.

Overlapping PCR was performed using two fragments obtained from theabove results as a template, and thereby 18 kinds of 1.4 kbppolynucleotides to be used as templates were obtained. The obtained genefragments were digested with a restriction enzyme, SpeI, ligated to thelinearized pDZ vector, which had already been digested with arestriction enzyme, XbaI, transformed into E. coli DH5α, and thetransformants were plated on a solid LB medium containing kanamycin (25mg/L).

The sequence information on the primers used for the preparation of thevector is shown in Table 8 below.

TABLE 8 SEQ ID NO Primer Sequence (5′ to 3′) 27 Spe1-impE1 164 1FGGGACTAGTGATTCCGGCCAACTGTCG 28 impE1 164-R 1RTGGGGCGCGTTGGCGTTCAGGATGCTC 29 impE1 164-H 1RTGGGGCGCGTTGGTGTTCAGGATGCTC 30 impE1 164-D 1RTGGGGCGCGTTGATCTTCAGGATGCTC 31 impE1 164-S 1RTGGGGCGCGTTGGGATTCAGGATGCTC 32 impE1 164-T 1RTGGGGCGCGTTGGGTTTCAGGATGCTC 33 impE1 164-N 1RTGGGGCGCGTTGGTTTTCAGGATGCTC 34 impE1 164-Q 1RTGGGGCGCGTTGCTGTTCAGGATGCTC 35 impE1 164-C 1RTGGGGCGCGTTGGCATTCAGGATGCTC 36 impE1 164-G 1RTGGGGCGCGTTGGCCTTCAGGATGCTC 37 impE1 164-P 1RTGGGGCGCGTTGCGGTTCAGGATGCTC 38 impE1 164-A 1RTGGGGCGCGTTGGGCTTCAGGATGCTC 39 impE1 164-V 1RTGGGGCGCGTTGGACTTCAGGATGCTC 40 impE1 164-I 1RTGGGGCGCGTTGGATTTCAGGATGCTC 41 impE1 164-L 1RTGGGGCGCGTTGCAGTTCAGGATGCTC 42 impE1 164-M 1RTGGGGCGCGTTGCATTTCAGGATGCTC 43 impE1 164-F 1RTGGGGCGCGTTGGAATTCAGGATGCTC 44 impE1 164-Y 1RTGGGGCGCGTTGGTATTCAGGATGCTC 45 impE1 164-W 1RTGGGGCGCGTTGCCATTCAGGATGCTC 46 Spe1-impE1 164 2RGGGACTAGTCATGAACTTGCCGCGCTC 47 impE1 164-R 2FGAGCATCCTGAACGCCAACGCGCCCCA 48 impE1 164-H 2FGAGCATCCTGAACACCAACGCGCCCCA 49 impE1 164-D 2FGAGCATCCTGAAGATCAACGCGCCCCA 50 impE1 164-S 2FGAGCATCCTGAATCCCAACGCGCCCCA 51 impE1 164-T 2FGAGCATCCTGAAACCCAACGCGCCCCA 52 impE1 164-N 2FGAGCATCCTGAAAACCAACGCGCCCCA 53 impE1 164-Q 2FGAGCATCCTGAACAGCAACGCGCCCCA 54 impE1 164-C 2FGAGCATCCTGAATGCCAACGCGCCCCA 55 impE1 164-G 2FGAGCATCCTGAAGGCCAACGCGCCCCA 56 impE1 164-P 2FGAGCATCCTGAACCGCAACGCGCCCCA 57 impE1 164-A 2FGAGCATCCTGAAGCCCAACGCGCCCCA 58 impE1 164-V 2FGAGCATCCTGAAGTCCAACGCGCCCCA 59 impE1 164-I 2FGAGCATCCTGAAATCCAACGCGCCCCA 60 impE1 164-L 2FGAGCATCCTGAACTGCAACGCGCCCCA 61 impE1 164-M 2FGAGCATCCTGAAATGCAACGCGCCCCA 62 impE1 164-F 2FGAGCATCCTGAATTCCAACGCGCCCCA 63 impE1 164-Y 2FGAGCATCCTGAATACCAACGCGCCCCA 64 impE1 164-W 2FGAGCATCCTGAATGGCAACGCGCCCCA

After selecting by PCR the colonies transformed with the vector intowhich the target gene was inserted, the plasmids were obtained using aconventionally known plasmid extraction method. The information on theobtained plasmids is shown in Table 9 below.

TABLE 9 No. Plasmid 1 pDZ-impE1 164R 2 pDZ-impE1 164H 3 pDZ-impE1 164D 4pDZ-impE1 164S 5 pDZ-impE1 164T 6 pDZ-impE1 164N 7 pDZ-impE1 164Q 8pDZ-impE1 164C 9 pDZ-impE1 164G 10 pDZ-impE1 164P 11 pDZ-impE1 164A 12pDZ-impE1 164V 13 pDZ-impE1 164I 14 pDZ-impE1 164L 15 pDZ-impE1 164M 16pDZ-impE1 164F 17 pDZ-impE1 164Y 18 pDZ-impE1 164W

Secondly, the procedure of preparing the vector for the introduction ofthe ImpE2(V2I) is as follows.

Based on the reported polynucleotide sequences, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template along with primer pairs between theprimer of SEQ ID NO: 65 and each of SEQ ID NOS: 66 to 83. PCR wasperformed by initial denaturation at 94° C. for 5 minutes; 20 cyclesconsisting of denaturation at 94° C. for 30 seconds, annealing at 55° C.for 30 seconds, and polymerization at 72° C. for 1 minute; and finalpolymerization at 72° C. for 5 minutes. As a result, 18 kinds of 0.7 kbppolynucleotides were obtained. Then, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template along with primer pairs between theprimer of SEQ ID NO: 84 and each of SEQ ID NOS: 85 to 102. PCR wasperformed by initial denaturation at 94° C. for 5 minutes; 20 cyclesconsisting of denaturation at 94° C. for 30 seconds, annealing at 55° C.for 30 seconds, and polymerization at 72° C. for 1 minute; and finalpolymerization at 72° C. for 5 minutes. As a result, 18 kinds of 0.7 kbppolynucleotides were obtained.

Overlapping PCR was performed using two fragments obtained from theabove results as a template, and thereby 18 kinds of 1.4 kbppolynucleotides to be used as templates were obtained. The obtained genefragments were digested with a restriction enzyme, XbaI, ligated to thelinearized pDZ vector, which had already been digested with arestriction enzyme, XbaI, transformed into E. coli DH5α, and thetransformants were plated on a solid LB medium containing kanamycin (25mg/L).

The sequence information on the primers used for the preparation of thevector is shown in Table 10 below.

TABLE 10 SEQ ID NO Primer Sequence (5′ to 3′) 65 XbaI-impE2 2 1FGGGTCTAGATTGCATGCTGTGCAAGA 66 impE2 2-R 1R GGAGTTTTTAGCGCGCATTCCAGTCCT67 impE2 2-H 1R GGAGTTTTTAGCGTGCATTCCAGTCCT 68 impE2 2-K 1RGGAGTTTTTAGCCTTCATTCCAGTCCT 69 impE2 2-D 1R GGAGTTTTTAGCGTCCATTCCAGTCCT70 impE2 2-E 1R GGAGTTTTTAGCTTCCATTCCAGTCCT 71 impE2 2-S 1RGGAGTTTTTAGCGGACATTCCAGTCCT 72 impE2 2-T 1R GGAGTTTTTAGCGGTCATTCCAGTCCT73 impE2 2-N 1R GGAGTTTTTAGCGTTCATTCCAGTCCT 74 impE2 2-Q 1RGGAGTTTTTAGCCTGCATTCCAGTCCT 75 impE2 2-C 1R GGAGTTTTTAGCGCACATTCCAGTCCT76 impE2 2-G 1R GGAGTTTTTAGCGCCCATTCCAGTCCT 77 impE2 2-P 1RGGAGTTTTTAGCTGGCATTCCAGTCCT 78 impE2 2-A 1R GGAGTTTTTAGCAGCCATTCCAGTCCT79 impE2 2-L 1R GGAGTTTTTAGCCAGCATTCCAGTCCT 80 impE2 2-M 1RGGAGTTTTTAGCCATCATTCCAGTCCT 81 impE2 2-F 1R GGAGTTTTTAGCGAACATTCCAGTCCT82 impE2 2-Y 1R GGAGTTTTTAGCGTACATTCCAGTCCT 83 impE2 2-W 1RGGAGTTTTTAGCCCACATTCCAGTCCT 84 XbaI-impE2 2 2RGGGTCTAGATTGCTCGCCCACGCGCA 85 impE2 2-R 2F AGGACTGGAATGCGCGCTAAAAACTCC86 impE2 2-H 2F AGGACTGGAATGCACGCTAAAAACTCC 87 impE2 2-K 2FAGGACTGGAATGAAGGCTAAAAACTCC 88 impE2 2-D 2F AGGACTGGAATGGACGCTAAAAACTCC89 impE2 2-E 2F AGGACTGGAATGGAAGCTAAAAACTCC 90 impE2 2-S 2FAGGACTGGAATGTCCGCTAAAAACTCC 91 impE2 2-T 2F AGGACTGGAATGACCGCTAAAAACTCC92 impE2 2-N 2F AGGACTGGAATGAACGCTAAAAACTCC 93 impE2 2-Q 2FAGGACTGGAATGCAGGCTAAAAACTCC 94 impE2 2-C 2F AGGACTGGAATGTGCGCTAAAAACTCC95 impE2 2-G 2F AGGACTGGAATGGGCGCTAAAAACTCC 96 impE2 2-P 2FAGGACTGGAATGCCAGCTAAAAACTCC 97 impE2 2-A 2F AGGACTGGAATGGCTGCTAAAAACTCC98 impE2 2-L 2F AGGACTGGAATGCTGGCTAAAAACTCC 99 impE2 2-M 2FAGGACTGGAATGATGGCTAAAAACTCC 100 impE2 2-F 2F AGGACTGGAATGTTCGCTAAAAACTCC101 impE2 2-Y 2F AGGACTGGAATGTACGCTAAAAACTCC 102 impE2 2-W 2FAGGACTGGAATGTGGGCTAAAAACTCC

After selecting by PCR the colonies transformed with the vector intowhich the target gene was inserted, the plasmids were obtained using aconventionally known plasmid extraction method. The information on theobtained plasmids is shown in Table 11 below.

TABLE 11 No. Plasmid 1 pDZ-impE2 2R 2 pDZ-impE2 2H 3 pDZ-impE2 2K 4pDZ-impE2 2D 5 pDZ-impE2 2E 6 pDZ-impE2 2S 7 pDZ-impE2 2T 8 pDZ-impE2 2N9 pDZ-impE2 2Q 10 pDZ-impE2 2C 11 pDZ-impE2 2G 12 pDZ-impE2 2P 13pDZ-impE2 2A 14 pDZ-impE2 2L 15 pDZ-impE2 2M 16 pDZ-impE2 2F 17pDZ-impE2 2Y 18 pDZ-impE2 2W

Lastly, the procedure of preparing the vector for the introduction ofthe ImpE2(G64E) is as follows.

Based on the reported polynucleotide sequences, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template denaturation at 94° C. for 30 seconds,annealing at 55° C. for 30 seconds, and poly along with primer pairsbetween the primer of SEQ ID NO: 103 and each of SEQ ID NOS: 104 to 121.PCR was performed by initial denaturation at 94° C. for 5 minutes; 20cycles consisting of denaturation at 94° C. for 30 seconds, annealing at55° C. for 30 seconds, and polymerization at 72° C. for 1 minute; andfinal polymerization at 72° C. for 5 minutes. As a result, 18 kinds of 1kbp polynucleotides were obtained. Then, the chromosomal genes ofCorynebacterium stationis CJI0323 were isolated, and gene fragments wereobtained by performing PCR using the chromosomal DNA of Corynebacteriumstationis CJI0323 as a template along with primer pairs between theprimer of SEQ ID NO: 84 and each of SEQ ID NOS: 85 to 102. PCR wasperformed by initial denaturation at 94° C. for 5 minutes; 20 cyclesconsisting of polymerization at 72° C. for 1 minute; and finalpolymerization at 72° C. for 5 minutes. As a result, 18 kinds of 1 kbppolynucleotides were obtained.

Overlapping PCR was performed using two fragments obtained from theabove results as a template, and thereby 18 kinds of 2 kbppolynucleotides to be used as templates were obtained. The obtained genefragments were digested with a restriction enzyme, XbaI, ligated to thelinearized pDZ vector, which had already been digested with arestriction enzyme, XbaI, transformed into E. coli DH5α, and thetransformants were plated on a solid LB medium containing kanamycin (25mg/L).

The sequence information on the primers used for the preparation of thevector is shown in Table 12 below.

TABLE 12 SEQ ID NO Primer Sequence (5′ to 3′) 103 XbaI-impE2 64 1FGGGTCTAGAAAAGAGCTTAAGGCAGCT GCT 104 impE2 64-R 1RGAAAATCATCTGGCGCAAAGAGCTCAT 105 impE2 64-H 1RGAAAATCATCTGGTGCAAAGAGCTCAT 106 impE2 64-D 1RGAAAATCATCTGGTCCAAAGAGCTCAT 107 impE2 64-K 1RGAAAATCATCTGCTTCAAAGAGCTCAT 108 impE2 64-S 1RGAAAATCATCTGGGACAAAGAGCTCAT 109 impE2 64-T 1RGAAAATCATCTGGGTCAAAGAGCTCAT 110 impE2 64-N 1RGAAAATCATCTGGTTCAAAGAGCTCAT 111 impE2 64-Q 1RGAAAATCATCTGCTGCAAAGAGCTCAT 112 impE2 64-C 1RGAAAATCATCTGGCACAAAGAGCTCAT 113 impE2 64-P 1RGAAAATCATCTGTGGCAAAGAGCTCAT 114 impE2 64-A 1RGAAAATCATCTGAGCCAAAGAGCTCAT 115 impE2 64-V 1RGAAAATCATCTGGACCAAAGAGCTCAT 116 impE2 64-I 1RGAAAATCATCTGGATCAAAGAGCTCAT 117 impE2 64-L 1RGAAAATCATCTGCAGCAAAGAGCTCAT 118 impE2 64-M 1RGAAAATCATCTGCATCAAAGAGCTCAT 119 impE2 64-F 1RGAAAATCATCTGGAACAAAGAGCTCAT 120 impE2 64-Y 1RGAAAATCATCTGGTACAAAGAGCTCAT 121 impE2 64-W 1RGAAAATCATCTGCCACAAAGAGCTCAT 122 XbaI-impE2 64 2RGGGTCTAGACGGTCAATGAAGTCTCAA CGG 123 impE2 64-R 2FATGAGCTCTTTGCGCCAGATGATTTTC 124 impE2 64-H 2FATGAGCTCTTTGCACCAGATGATTTTC 125 impE2 64-D 2FATGAGCTCTTTGGACCAGATGATTTTC 126 impE2 64-K 2FATGAGCTCTTTGAAGCAGATGATTTTC 127 impE2 64-S 2FATGAGCTCTTTGTCCCAGATGATTTTC 128 impE2 64-T 2FATGAGCTCTTTGACCCAGATGATTTTC 129 impE2 64-N 2FATGAGCTCTTTGAACCAGATGATTTTC 130 impE2 64-Q 2FATGAGCTCTTTGCAGCAGATGATTTTC 131 impE2 64-C 2FATGAGCTCTTTGTGCCAGATGATTTTC 132 impE2 64-P 2FATGAGCTCTTTGCCACAGATGATTTTC 133 impE2 64-A 2FATGAGCTCTTTGGCTCAGATGATTTTC 134 impE2 64-V 2FATGAGCTCTTTGGTCCAGATGATTTTC 135 impE2 64-I 2FATGAGCTCTTTGATCCAGATGATTTTC 136 impE2 64-L 2FATGAGCTCTTTGCTGCAGATGATTTTC 137 impE2 64-M 2FATGAGCTCTTTGATGCAGATGATTTTC 138 impE2 64-F 2FATGAGCTCTTTGTTCCAGATGATTTTC 139 impE2 64-Y 2FATGAGCTCTTTGTACCAGATGATTTTC 140 impE2 64-W 2FATGAGCTCTTTGTGGCAGATGATTTTC

After selecting by PCR the colonies transformed with the vector intowhich the target gene was inserted, the plasmids were obtained using aconventionally known plasmid extraction method. The information on theobtained plasmids is shown in Table 13 below.

TABLE 13 No. Plasmid 1 pDZ-impE2 64R 2 pDZ-impE2 64H 3 pDZ-impE2 64D 4pDZ-impE2 64K 5 pDZ-impE2 64S 6 pDZ-impE2 64T 7 pDZ-impE2 64N 8pDZ-impE2 64Q 9 pDZ-impE2 64C 10 pDZ-impE2 64P 11 pDZ-impE2 64A 12pDZ-impE2 64V 13 pDZ-impE2 64I 14 pDZ-impE2 64L 15 pDZ-impE2 64M 16pDZ-impE2 64F 17 pDZ-impE2 64Y 18 pDZ-impE2 64W

Example 5-2: Preparation of Strains where Amino Acids at Positions ofModified Products (ImpE1, ImpE2) are Substituted with Another AminoAcids, and Comparison of Ability to Produce IMP

The 54 kinds of plasmids prepared in Example 5-1 were transformed intothe CJI0323 strain. The strains in which the vector was inserted intothe chromosome by recombination of the homologous sequences wereselected on a medium containing kanamycin (25 mg/L). The selectedprimary strains were subjected to a second cross-over. The introductionof the modification in the finally transformed strains was confirmed byperforming PCR using the primer pair of SEQ ID NOS: 15 and 16, followedby nucleotide sequencing analysis. The strain names according to theinserted modifications are shown in Table 14 below.

TABLE 14 No. Strain 1 CJI0323::impE1(E164R) 2 CJI0323::impE1(E164H) 3CJI0323::impE1(E164D) 4 CJI0323::impE1(E164S) 5 CJI0323::impE1(E164T) 6CJI0323::impE1(E164N) 7 CJI0323::impE1(E164Q) 8 CJI0323::impE1(E164C) 9CJI0323::impE1(E164G) 10 CJI0323::impE1(E164P) 11 CJI0323::impE1(E164A)12 CJI0323::impE1(E164V) 13 CJI0323::impE1(E1641) 14CJI0323::impE1(E164L) 15 CJI0323::impE1(E164M) 16 CJI0323::impE1(E164F)17 CJI0323::impE1(E164Y) 18 CJI0323::impE1(E164W) 19 CJI0323::impE2(V2R)20 CJI0323::impE2(V2H) 21 CJI0323::impE2(V2K) 22 CJI0323::impE2(V2D) 23CJI0323::impE2(V2E) 24 CJI0323::impE2(V2S) 25 CJI0323::impE2(V2T) 26CJI0323::impE2(V2N) 27 CJI0323::impE2(V2Q) 28 CJI0323::impE2(V2C) 29CJI0323::impE2(V2G) 30 CJI0323::impE2(V2P) 31 CJI0323::impE2(V2A) 32CJI0323::impE2(V2L) 33 CJI0323::impE2(V2M) 34 CJI0323::impE2(V2F) 35CJI0323::impE2(V2Y) 36 CJI0323::impE2(V2W) 37 CJI0323::impE2(G64R) 38CJI0323::impE2(G64H) 39 CJI0323:impE2(G64D) 40 CJI0323::impE2(G64K) 41CJI0323::impE2(G64S) 42 CJI0323::impE2(G64T) 43 CJI0323::impE2(G64N) 44CJI0323::impE2(G64Q) 45 CJI0323::impE2(G64Q 46 CJI0323::impE2(G64P) 47CJI0323::impE2(G64A) 48 CJI0323::impE2(G64V) 49 CJI0323::impE2(G64I) 50CJI0323:impE2(G64L) 51 CJI0323::impE2(G64M) 52 CJI0323::impE2(G64F) 53CJI0323:impE2(G64Y) 54 CJI0323::impE2(G64W)

The cultivation was performed in the same manner as in Example 1 and theconcentration of IMP produced thereof was analyzed (Table 15).

TABLE 15 Concentration (g/L) of IMP production in strains with combinedintroduction of impE1, impE2 modifications Strain Average IMP ControlCJI0323_impE1E2(WT) 2.32 1 CJI0323::impE1(E164R) 9.42 2CJI0323::impE1(E164H) 8.47 3 CJI0323::impE1(E164D) 7.37 4CJI0323::impE1(E164S) 8.56 5 CJI0323::impE1(E164T) 8.85 6CJI0323::impE1(E164N) 9.13 7 CJI0323::impE1(E164Q) 7.45 8CJI0323::impE1(E164C) 7.37 9 CJI0323::impE1(E164G) 9.13 10CJI0323::impE1(E164P) 9.43 11 CJI0323::impE1(E164A) 7.44 12CJI0323::impE1(E164V) 8.18 13 CJI0323::impE1(E1641) 8.09 14CJI0323::impE1(E164L) 7.85 15 CJI0323::impE1(E164M) 7.39 16CJI0323::impE1(E164F) 7.56 17 CJI0323::impE1(E164Y) 7.60 18CJI0323::impE1(E164W) 8.56 19 CJI0323::impE2(V2R) 7.99 20CJI0323::impE2(V2H) 8.75 21 CJI0323::impE2(V2K) 8.66 22CJI0323::impE2(V2D) 8.28 23 CJI0323::impE2(V2E) 9.32 24CJI0323::impE2(V2S) 6.37 25 CJI0323::impE2(V2T) 8.37 26CJI0323::impE2(V2N) 9.80 27 CJI0323::impE2(V2Q) 7.04 28CJI0323::impE2(V2C) 7.23 29 CJI0323::impE2(V2G) 7.71 30CJI0323::impE2(V2P) 7.80 31 CJI0323::impE2(V2A) 6.57 32CJI0323::impE2(V2L) 6.42 33 CJI0323::impE2(V2M) 9.20 34CJI0323::impE2(V2F) 9.43 35 CJI0323::impE2(V2Y) 8.37 36CJI0323::impE2(V2W) 7.22 37 CJI0323::impE2(G64R) 4.42 38CJI0323::impE2(G64H) 5.14 39 CJI0323::impE2(G64D) 11.53 40CJI0323::impE2(G64K) 4.8 41 CJI0323::impE2(G64S) 5.7 42CJI0323::impE2(G64T) 5.52 43 CJI0323::impE2(G64N) 5.9 44CJI0323::impE2(G64Q) 4.8 45 CJI0323::impE2(G64C) 5.9 46CJI0323::impE2(G64P) 4.75 47 CJI0323::impE2(G64A) 4.58 48CJI0323::impE2(G64V) 4.56 49 CJI0323::impE2(G64I) 5.89 50CJI0323::impE2(G64L) 5.6 51 CJI0323::impE2(G64M) 4.3 52CJI0323::impE2(G64F) 5.89 53 CJI0323::impE2(G64Y) 4.6 54CJI0323::impE2(G64W) 4.76

As shown above, all of the modified strains showed an increase in theability to produce IMP compared to each of the control strains, andthus, it was confirmed that the three positions of modification areimportant sites that have a significant effect on the increase of theability of the ImpE protein with respect to IMP export.

Example 6: Introduction of impE1, impE2 Modifications Based onIMP-Producing Strains Example 6-1: Preparation of Strains with impE1,impE2 Modifications Based on IMP-Producing Strains

To confirm the effect of introduction of impE1 and impE2 modifications,An IMP-producing strain was prepared in which the activities ofadenylosuccinate synthetase and IMP dehydrogenase corresponding to thedegradation pathway of IMP in the ATCC6872 strain were attenuated. Theinitiation codon was changed by changing the first base from ‘a’ to ‘t’in each nucleotide sequence of the two genes purA and guaB, which encodethe two enzymes. The strain in which the expression of the two genes wasattenuated in the ATCC6872 strain was named CJI9088. ThepDZ-impE1(E164K), pDZ-impE2(V2I), and pDZ-impE2(G64E) vectors preparedin Example 4-2 were transformed into the CJI9088 strain byelectroporation, and the pDZ-impE2(G64D) vector prepared in Example 5-1was transformed into the CJI9088_impE1(E164K)_impE2(V2I) strain byelectroporation. The strains in which the vectors were inserted into thechromosome by recombination of the homologous sequences were selected ona medium containing kanamycin (25 mg/L). The selected primary strainswere subjected to a second cross-over. The introduction of themodification in the finally transformed strains was confirmed byperforming PCR using the primer pair of SEQ ID NOS: 15 and 16, followedby nucleotide sequencing analysis.

The ability of the prepared strains (i.e., CJI9088, CJI9088_impE1(E164K), CJI9088_impE2(V2I), CJI9088_impE2(G64E), andCJI9088_impE1(E164K)_impE2(V2I)(G64D)) to produce IMP was evaluated.Upon completion of the culture, the amount of IMP production wasmeasured by HPLC and the results are shown in Table 16 below.

TABLE 16 Strain IMP (g/L) CJI9088 0.52 CJI9088_impE1(E164K) 0.84CJI9088_impE2(V2I) 0.93 CJI9088_impE2(G64E) 1.73CJI9088_impE1(E164K)_impE2(V2I)(G64D) 4.30

Upon confirming the amount of IMP accumulated in the culture medium, itwas confirmed that these strains showed an increase of IMP production byat least 61%, and a maximum increase of 727%, compared to the parentstrain, CJ9088. Accordingly, the increase in the amount of IlVIPproduction due to modifications of the ImpE protein of the presentdisclosure can be interpreted to be very meaningful.

From the foregoing, a skilled person in the art to which the presentdisclosure pertains will be able to understand that the presentdisclosure may be embodied in other specific forms without modifying thetechnical concepts or essential characteristics of the presentdisclosure. In this regard, the exemplary embodiments disclosed hereinare only for illustrative purposes and should not be construed aslimiting the scope of the present disclosure. On the contrary, thepresent disclosure is intended to cover not only the exemplaryembodiments but also various alternatives, modifications, equivalents,and other embodiments that may be included within the spirit and scopeof the present disclosure as defined by the appended claims.

The invention claimed is:
 1. A protein variant exporting 5′-inosinemonophosphate, wherein, in the amino acid sequence of SEQ ID NO: 2, (i)the 2^(nd) amino acid, (ii) the 64^(th) amino acid, or (iii) the 2^(nd)amino acid and the 64^(th) amino acid are each substituted with anotheramino acid.
 2. The protein variant according to claim 1, wherein the2^(nd) amino acid is substituted with an amino acid selected from thegroup consisting of isoleucine, phenylalanine, methionine, glutamicacid, histidine, and asparagine; (ii) the 64^(th) amino acid issubstituted with an amino acid selected from the group consisting ofaspartate, glutamic acid, asparagine, cysteine, isoleucine, andphenylalanine; or (iii) the 2^(nd) amino acid and the 64^(th) amino acidare each substituted with an amino acid selected from the groupconsisting of methionine, glutamic acid, histidine, asparagine,aspartate, cysteine, isoleucine, and phenylalanine.